The impact of p53 on numerous aspects of normal and pathophysiological contexts is apparent from both in vitro and in vivo studies. Literature supports p53 is a critical regulator of cell cycle inhibition, genomic stability and apoptosis inclusive of most cell and tissue types. It is clear that p53 functions as a transcription factor to regulate the above effects and combined structure/mutation studies demonstrate the DNA binding and proline-rich domains of p53 are crucial to many of its protective effects in vertebrates. However, increasing evidence suggests p53 also controls apoptosis in a transcription-independent route. The goal of this research proposal is to explore the central mechanisms by which p53 can induce apoptosis in a transcription-independent manner using several novel in vitro and in vivo models. This work will be done in a unique cell-free system developed by our laboratory, several cell lines of diverse origin, and in primary cells from a recently generated knock-in mouse. Efforts will focus on the role of pro-apoptotic Bcl-2 family members in the permeabilization of the mitochondrial outer membrane and how p53 regulates this process.